Pancreatic ductal adenocarcinoma remains one of the most lethal malignancies with only an 11% 5-year survival rate. Oncogenic microRNAs (miRNA), particularly miR-21, miR-155, and miR-18a, drive tumor progression by silen...Pancreatic ductal adenocarcinoma remains one of the most lethal malignancies with only an 11% 5-year survival rate. Oncogenic microRNAs (miRNA), particularly miR-21, miR-155, and miR-18a, drive tumor progression by silencing tumor suppressors and promoting chemoresistance. Single-target miRNA inhibition has shown limited clinical efficacy because of complex network redundancy and compensatory pathway activation, necessitating multitarget therapeutic approaches. We designed and validated a trispecific miRNA sponge construct containing high-affinity target sites for all 3 oncomiRs, demonstrated in silico through miRNAsong analysis with an approximately 11-kcal/mol thermodynamic specificity gap relative to off-targets. The sponge was functionally tested in the human pancreatic ductal adenocarcinoma cell lines PANC-1 and AsPC-1 using comprehensive assays including dual-luciferase reporter, quantitative reverse transcription polymerase chain reaction, flow cytometry-based apoptosis analysis, scratch-wound migration, and gemcitabine chemosensitization studies. In AsPC-1 cells, the trispecific sponge achieved 99.0%-99.9% silencing of target miRNAs and 349-fold reporter reduction, inducing a 6.1-fold increase in apoptosis and approximately 44% reduction in wound closure at 72 hours compared with nontargeting controls. PANC-1 cells showed moderate but significant responses with 65%-98% miRNA silencing, a 2.2-fold increase in apoptosis, and approximately 51% reduction in wound closure at 72 hours under identical assay conditions. Mechanistically, simultaneous miRNA inhibition synergistically reactivated tumor suppressor genes PDCD4, ESR1, and NOTCH2 (6.3-7.7-fold upregulation) and sensitized chemoresistant cells to gemcitabine by 1.5- to 1.8-fold. Across the evaluated functional endpoints, the trispecific sponge conferred approximately 1.2- to 95-fold changes relative to the nontargeting control and, in many instances, elicited equal or greater effects than single-target constructs, consistent with a broader network-level impact rather than uniform superiority in every assay. This platform represents a promising strategy for pancreatic ductal adenocarcinoma therapy warranting preclinical development and clinical translation. SIGNIFICANCE STATEMENT: Pancreatic cancer chemoresistance drives poor survival. The trispecific microRNA sponge simultaneously targets miR-21, miR-155, and miR-18a, synergistically reactivating tumor suppressors and enhancing gemcitabine efficacy more than single-target approaches. This multitarget microRNA strategy represents a novel therapeutic platform for overcoming chemoresistance in pancreatic cancer.
J Pharmacol Exp Ther
· 2026 May · PMID 42019163
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Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. The...Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. The absence of molecular targets in TNBC limits treatment options and contributes to increased rates of recurrence, metastasis, and resistance to conventional therapies. TNBC, however, is rich in tumor-infiltrating lymphocytes; hence, elevated programmed death-ligand 1 expression makes these tumors amenable to immunotherapy. A variety of antibodies and immunomodulatory drugs are being explored for TNBC treatment, this review specifically focuses on design of immunomodulatory peptides for TNBC treatment. This review comprehensively discusses the peptide-based approaches for immunomodulating tumor microenvironment (TME) of TNBC and to enhance antitumor immune response. The peptide-mediated modulation of innate immune cells including tumor-associated macrophages, neutrophils, dendritic cells, and natural killer cells, as well as T cells of the adaptive immune system is explored in detail. The applications of peptides as immune checkpoint inhibitors and highlights of emerging strategies that employ peptides to induce immunogenic cell death to stimulate antitumor immunity are discussed. Immunogenic cell death inducing peptides promote the release of immunogenic signals such as damage-associated molecular patterns from dying cancer cells, which further activate dendritic cells, T cells, and neutrophils, thereby reshaping the TME to support robust antitumor immunity. These strategies underscore the transformative potential of peptide therapeutics to harness the immune system and reshape TME, offering a promising avenue for more effective and durable TNBC treatment. SIGNIFICANCE STATEMENT: With growing interest in peptides as tumor microenvironment modulator, this review provides an in-depth analysis of interactions and crosstalk between immune and tumor cells and explores therapeutic potential of peptides in modulating immune cell signaling pathways, with ultimate impact as anticancer agents for triple-negative breast cancer.
Age is a prominent risk factor for the development of kidney disease, emphasizing the importance of understanding and mitigating the mechanisms underlying renal aging. Aging mitochondria in tubular cells become increasin...Age is a prominent risk factor for the development of kidney disease, emphasizing the importance of understanding and mitigating the mechanisms underlying renal aging. Aging mitochondria in tubular cells become increasingly dysfunctional, reducing ATP production, and increasing reactive oxygen species, which drives inflammation. Pharmacological induction of mitochondrial biogenesis, the generation of new and functional mitochondria, restores renal function in mouse models of kidney disease, including acute kidney injury and diabetic kidney disease. The potential of this therapy in age-related renal decline remains unknown. 2,4,6-trifluoro-N-(6-(4-methylpiperazine-1-carbonyl)pyridin-2-yl)benzamide (MARY1) is a novel, selective serotonin 2B receptor antagonist that induces mitochondrial biogenesis and improves recovery after acute kidney injury in vivo. Young (12-week-old) and aged (104-week-old) male mice were treated with MARY1 (0.3 mg/kg, i.p.) or vehicle daily for 3 weeks. Mice were euthanized at 15- and 107-weeks, respectively, and the kidneys collected for analyses. Aged 107-week-old mice exhibited increased phosphorylation of mammalian target of rapamycin, decreased AMP-activated protein kinase phosphorylation, decreased ATP, and increased inflammation and fibrosis in the renal cortex compared with young controls. MARY1 treatment restored AMP-activated protein kinase phosphorylation, and increased protein kinase B activity and ATP content. MARY1 decreased the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 and reduced markers of renal inflammation and fibrosis in the aged kidney, including vascular cell adhesion molecule 1, collagen-1, and collagen deposition. These findings suggest that MARY1 treatment restores mitochondrial metabolism and ameliorates age-related renal inflammation and fibrosis in mice, supporting its potential as a therapeutic strategy to preserve renal function during aging. SIGNIFICANCE STATEMENT: Age-related kidney dysfunction is driven in part by declining mitochondrial metabolism and chronic inflammation. This study reveals that the novel selective serotonin 2B receptor antagonist 2,4,6-trifluoro-N-(6-(4-methylpiperazine-1-carbonyl)pyridin-2-yl)benzamide restores mitochondrial metabolism and reduces renal inflammation and fibrosis in aged mice, supporting pharmacological induction of mitochondrial biogenesis and 2,4,6-trifluoro-N-(6-(4-methylpiperazine-1-carbonyl)pyridin-2-yl)benzamide as a promising strategy to preserve kidney function during aging.
Neuroendocrine prostate cancer is an aggressive, therapy-resistant subtype of prostate cancer with unclear underlying molecular mechanisms and limited effective treatments. Through single-cell transcriptomic analysis, we...Neuroendocrine prostate cancer is an aggressive, therapy-resistant subtype of prostate cancer with unclear underlying molecular mechanisms and limited effective treatments. Through single-cell transcriptomic analysis, we found that SOX11 is specifically expressed in neuroendocrine cell populations. Further studies confirmed that SOX11 promotes neuroendocrine transdifferentiation in prostate cancer via CXCL10/CXCR3 activation, thereby enhancing the migration and invasion abilities of prostate cancer cells. These results suggest that SOX11 may play a central role in driving neuroendocrine prostate cancer progression, which is expected to serve as a candidate factor for subsequent therapeutic exploration. SIGNIFICANCE STATEMENT: This study reveals the mechanism by which the SOX11/CXCL10/CXCR3 axis promotes neuroendocrine transdifferentiation of prostate cancer, thereby identifying potential targets for the development of targeted therapeutic strategies against neuroendocrine prostate cancer.
Glioblastoma multiforme is the most commonly diagnosed type of brain tumor, with a poor prognosis and a high rate of recurrence. Because of its highly aggressive nature and the lack of efficient treatment options, novel...Glioblastoma multiforme is the most commonly diagnosed type of brain tumor, with a poor prognosis and a high rate of recurrence. Because of its highly aggressive nature and the lack of efficient treatment options, novel therapeutic strategies are needed. Ferroptosis is an iron-dependent, unique type of cell death, which provides an alternative way to eradicate cancer cells that are resistant to apoptosis and other cell death mechanisms. CP55-940 (CP) and WIN 55212-2 (WIN) are synthetic cannabinoid receptor agonists with various biological activities, including neuroprotective and anticancer effects; however, their mechanism of action has not been fully uncovered. In the present study, the potential of CP and WIN in glioblastoma cells was investigated. Cell viability was determined with the MTT assay. Labile iron pool and reactive oxygen species generation were visualized with confocal microscopy. Malondialdehyde assay was performed to detect lipid peroxidation. Gene expressions of ferroptotic hallmarks, glutathione peroxidase-4, and transferrin receptor 1 were determined by RT-qPCR. Protein expression levels of iron-responsive element-binding protein 2, solute carrier family 7 member 11, and glutathione peroxidase-4 were analyzed by western blotting. Results demonstrated that CP and WIN significantly induce ferroptotic pathways in glioblastoma cells via increased oxidative stress, labile iron pool, and lipid peroxidation. Furthermore, it was determined for the first time that both compounds significantly upregulate the transferrin receptor 1 gene expression. In conclusion, the present study demonstrated for the first time that cannabinoid derivatives CP and WIN alter iron regulation and initiate ferroptosis in glioblastoma cells, rendering them potential candidates in therapy. SIGNIFICANCE STATEMENT: We explored the ferroptotic activity of cannabinoid derivatives (CP and WIN) in glioblastoma cells for the first time. Additionally, we report for the first time that cannabinoid derivatives alter cellular iron levels, causing increased labile iron pool via upregulating the transferrin gene significantly.
J Pharmacol Exp Ther
· 2026 Apr · PMID 41945989
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Steady-state volume of distribution can be predicted using tissue-to-plasma partition coefficients (K) and tissue volumes. K values are important components of physiologically based pharmacokinetic models, allowing for e...Steady-state volume of distribution can be predicted using tissue-to-plasma partition coefficients (K) and tissue volumes. K values are important components of physiologically based pharmacokinetic models, allowing for estimation of distribution kinetics and simulation of concentration-time profiles. Many in silico approaches have been developed to predict tissue K values based on physicochemical processes that govern drug distribution. However, these methods frequently overpredict or underpredict tissue K values, highlighting the need to consider additional mechanisms that can impact drug distribution kinetics. Many drugs have been shown to bind to rat and human fatty acid binding proteins (FABPs) in vitro but the impact of this binding to drug distribution has not been incorporated into K predictions. We hypothesized that incorporating intracellular protein binding into tissue K predictions will improve K prediction accuracy. Using liver as a model organ, 4 physiologically based dynamic liver distribution models (LDMs) were developed to assess the role of distribution processes in K predictions. The developed LDMs incorporated known distribution mechanisms and intracellular drug binding to liver FABP (FABP1). The liver K values for drugs that bind to FABP1 were accurately predicted using the LDM that incorporates lipid partitioning, albumin distribution, and FABP1 binding but not using LDMs without FABP1 binding. Human FABP1 expression was quantified in 61 human livers and the interindividual variability in tissue FABP1 binding was incorporated into tissue K predictions. These simulations showed that intracellular FABP1 binding can cause interindividual variability in K values and result in higher cytosolic drug concentrations. SIGNIFICANCE STATEMENT: This study shows that incorporating intracellular protein binding such as binding to fatty acid binding protein 1 into tissue K predictions improves accuracy of the predictions. The novel dynamic liver distribution model can be extrapolated to other organs of interest and integrated into full-body physiologically based pharmacokinetic models to predict drug distribution kinetics. With dynamic and saturable distribution mechanisms incorporated into a physiologically based pharmacokinetic model, nonlinear distribution kinetics can be simulated for various drugs.
J Pharmacol Exp Ther
· 2026 Apr · PMID 41935399
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Black box warnings represent the most severe US Food and Drug Administration safety alerts. Currently, they are fragmented across individual product labels and not systematically accessible at the level of drugs or drug...Black box warnings represent the most severe US Food and Drug Administration safety alerts. Currently, they are fragmented across individual product labels and not systematically accessible at the level of drugs or drug classes. This study provides a comprehensive overview of drugs with black box warnings and identifies class- and category-specific patterns of adverse effects, aiming to support risk benefit assessments and rational treatment. Six hundred twenty-six drugs across 250 drug classes were identified as carrying black box warnings. Drug classes were unevenly distributed, with many classes represented by a single drug, whereas a small number of large classes accounted for a substantial proportion of entries, for example, μ-opioid receptor agonists, cyclooxygenase inhibitors, angiotensin-converting enzyme inhibitors and kinase inhibitors. Across all drugs, 1016 adverse effect category listings were identified, demonstrating that multiple serious risks are frequently combined within a single boxed warning. Cardiac and cardiovascular adverse effects are the most prevalent category (18.7%), followed by immunologic and allergic reactions (11.4%), beside drug category-specific patterns. The standardized adverse effect count ranged from 1.1 in endocrine system drugs to 2.1 in cytotoxic treatments, indicating marked differences in the density and variety of serious adverse effects between drug categories. Black box warnings reveal pronounced heterogeneity across drugs, but show consistent, mechanism related patterns at the level of drug classes and categories. These patterns allow estimation of probable, high-risk adverse effects and risk profiles. A structured, drug class-based overview of black box warnings can improve awareness of safety risks and support rational prescribing. SIGNIFICANCE STATEMENT: Black box warnings are the most severe US Food and Drug Administration safety alerts, but they are fragmented across individual product labels. By systematically analyzing US Food and Drug Administration boxed warnings across drugs, drug classes, and adverse effect categories, this study reveals pronounced heterogeneity across individual drugs, but consistent, mechanism related risk patterns, with cardiovascular and immunologic adverse effects being most prevalent. A structured, drug class-based overview enables identification of high-risk profiles, supporting risk benefit assessment and rational prescribing.
PD-1 and PD-L1 are programmed cell death proteins and ligands that form a key axis of immune checkpoints that tumors use to escape immune surveillance. Although immune checkpoint inhibitors that activate this pathway hav...PD-1 and PD-L1 are programmed cell death proteins and ligands that form a key axis of immune checkpoints that tumors use to escape immune surveillance. Although immune checkpoint inhibitors that activate this pathway have revolutionized the treatment of cancer, resistance and unpredictable responses to a patient are still significant issues. There is growing evidence that noncoding RNAs (ncRNAs) such as microRNAs, long noncoding RNAs, and circular RNAs are key regulators of PD-1/PD-L1 signaling. These ncRNAs control the PD-L1 expression by directing its mRNA and indirectly maintaining the upstream signaling processes, consequently influencing tumor progression, immune cell activity, and drug responses. This review, based on existing research on the mechanistic functions of ncRNAs in PD-1/PD-L1-based immune suppression, discusses the possibility of using ncRNAs as biomarkers to predict immunotherapy response and as new therapies. We also address opportunities in the field of translation, such as ncRNA-based interventions and combinations with checkpoint blockade, and the challenges, which require resolution to move to the clinical practice. The combination of ncRNA biology and tumor immunology has potential applications in the area of precision immunotherapy and the creation of more meaningful treatment tools against malignancies that are resistant to treatment. SIGNIFICANCE STATEMENT: The current review outlines the regulatory functions of noncoding RNAs in the programmed cell death protein 1/programmed cell death ligand 1 immune checkpoint and highlights their role in tumor immune evasion and therapeutic responses regulation. With an interwoven method of mechanistic understanding and translational approaches, this study outlines noncoding RNAs as potential biomarkers and therapeutic targets and provide new approaches to increment the activity of checkpoint blocks and improve precision cancer immunotherapy.
Alzheimer disease (AD) and aging have similar molecular mechanisms that are affected by genetic as well as environmental variables. Based on current research, gut microbiomes contribute to age-specific biological process...Alzheimer disease (AD) and aging have similar molecular mechanisms that are affected by genetic as well as environmental variables. Based on current research, gut microbiomes contribute to age-specific biological processes and play an essential role in maintaining host homeostasis. Several molecular processes, including the host DNA methylation mechanism, are affected by microbially derived metabolites such as short-chain fatty acids, folate, and choline. This interaction establishes a mechanistic causal relationship that further shapes gene expression, inflammatory balance, and neuronal function in aging and related diseases. In this review, we looked at recent research showing how gut dysbiosis and its associated metabolites impact DNA methylation, which consequently contributes to disease progression in AD and aging. We also talked about how the DNA clock and age-associated methylation drifts can be used for forecasting biological aging. In addition, we discussed recent findings on how microbial and diet-based interventions may restore the methylation patterns that might be involved in aging and neurodegenerative processes. We also implicated the possible use of methylation-based biomarkers in the diagnosis of AD. Additionally, we have also explored the potential therapeutic benefits of using microbiome modulators, dietary modifications, and pharmacological interventions. Next, we highlighted the importance of multiomics and longitudinal studies to build the causal connection underlying methylation dynamics and microbial changes in neurodegeneration. Altogether, this review highlights the potential of the microbiome-methylation axis as an approach to understanding aging and establishing precision strategies to maintain cognitive health. SIGNIFICANCE STATEMENT: This review explores the interplay between DNA methylation and gut microbiota in aging and Alzheimer's disease. It highlights the gut-brain axis and summarizes recent findings on microbiome-driven epigenetic changes and metabolites influencing cognitive decline. The review also emphasizes microbiome-targeted therapeutic strategies for age-related disorders. Overall, it integrates current molecular insights with emerging approaches for the detection, prevention, and management of Alzheimer's disease and associated cognitive challenges.
Fernandopulle NA, Ding J, Francis G
… +4 more, Hulett MD, Soeding PF, May LT, Mackay GA
J Pharmacol Exp Ther
· 2026 Apr · PMID 41886886
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Protamine is a polybasic drug commonly used in cardiac surgery to chemically inhibit, and thereby reverse, the anticoagulant effect of heparin. However, protamine can trigger severe anaphylaxis, with both IgE-dependent a...Protamine is a polybasic drug commonly used in cardiac surgery to chemically inhibit, and thereby reverse, the anticoagulant effect of heparin. However, protamine can trigger severe anaphylaxis, with both IgE-dependent and IgE-independent mechanisms implicated. Recent research has linked the receptor MRGPRX2 (Mas-related G protein-coupled receptor X2) to IgE-independent mast cell activation by polybasic drugs, but its direct interaction with protamine has not been studied. This study investigated the role of MRGPRX2 in protamine-induced mast cell activation and explored how heparin influenced MRGPRX2 activation by protamine and other MRGPRX2 agonists. We used an in vitro cultured human mast cell line, Laboratory of Allergic Diseases 2, that constitutively expresses MRGPRX2. Receptor dependence of agonist-mediated signal transduction was demonstrated using a CRISPR MRGPRX2 knock down Laboratory of Allergic Diseases 2 cell line and a MRGPRX2 inverse-agonist. Mast cell activation was quantified using intracellular calcium mobilization, cellular degranulation, and cytokine release. Protamine-induced a concentration-dependent mast cell activation and was characterized as an unbiased partial agonist at MRGPRX2. Protamine-induced responses were significantly reduced by MRGPRX2 knock down and receptor antagonism. Heparin, which is known to complex with protamine, reduced the mast cell activation induced by protamine, compound 48/80, and LL-37, but not by the other MRGPRX2 agonists assessed. Isothermal titration calorimetry demonstrated that this selectivity relates to ligand ability to bind heparin. Recapitulation of clinically used ratios of protamine:heparin revealed that under these conditions, protamine still triggered effective mast cell activation, highlighting the need for further research to better understand the clinical relevance of MRGPRX2 activation by protamine. SIGNIFICANCE STATEMENT: This study identified protamine, a polybasic drug commonly used in cardiac surgery, as a partial agonist at the MRGPRX2 receptor, and that heparin significantly inhibited the receptor activation by protamine and other endogenous MRGPRX2 agonists. However, the commonly used clinical ratio of protamine:heparin still permitted mast cell activation in vitro.
Aging is a progressive process of multisystem physiological function decline driven by multiple factors, NAD is a potential compound for alleviating senescence. But the biological effects and mechanism of NAD against agi...Aging is a progressive process of multisystem physiological function decline driven by multiple factors, NAD is a potential compound for alleviating senescence. But the biological effects and mechanism of NAD against aging and aging-associated diseases are not fully elucidated. In this study, we first evaluated the protective effect of NAD on senescent cells, and then evaluated the blocking effect of aging and aging-associated pneumonia in vivo. The results showed that NAD could significantly improve the cell survival rate and oxidative stress, reduced the expression of aging markers and inflammatory factors. In animal experiments, NAD significantly improved the cognitive function of D-galactose induced aging mice by promoting the expression of BDNF, SYN and PSD-95. In addition, NAD can significantly enhance the activities of antioxidant enzymes, and reduce the release of proinflammatory cytokines. Quantitative real-time reverse transcription polymerase chain reaction results further showed that NAD could attenuate senescence induced cell cycle arrest by downregulating the expression of senescence-related genes such as p53, p21, and p16. Based on the aging mouse model induced by D-galactose, the acute pneumonia model was induced by Acinetobacter baumannii, a clinically relevant opportunistic pathogen. The results showed that the mice treated with NAD showed stronger resistance to pneumonia infection, the amount of bacterial colonization in the lungs decreased significantly and the weight loss was significantly improved. In terms of immune response, NAD improved the symptoms of pneumonia by enhancing humoral immunity and regulating cellular immunity. Therefore, as an effective agent for mitigating oxidative senescence, NAD holds potential as a dietary supplement to enhance health-span. SIGNIFICANCE STATEMENT: This study found that NAD can not only significantly improve the antioxidant capacity and cognitive function of aging mice, but also effectively enhance the immune defense of aging mice against bacterial infection, which provided experimental basis for its application in the prevention and treatment of aging and concurrent diseases.
Given the high prevalence of polypharmacy among patients with depression, this study systematically assesses the drug-drug interaction (DDI) risks of the investigational antidepressant ammoxetine (AMT) mediated by cytoch...Given the high prevalence of polypharmacy among patients with depression, this study systematically assesses the drug-drug interaction (DDI) risks of the investigational antidepressant ammoxetine (AMT) mediated by cytochrome P450 2D6 (CYP2D6) (metabolic enzyme) and multidrug and toxin extrusion protein 1 (MATE1) (renal transporter), and characterizes the resulting metabolism-transport interaction cascade. In vitro DDI risk screening for AMT was performed using human liver microsomes, recombinant CYP enzymes, and transporter-overexpressing cell lines. Static mechanistic models were used to predict human DDI risks, with sensitivity analyses incorporated to assess CYP2D6 poor metabolizers (PMs). Finally, in vivo validation was carried out in Institute of Cancer Research mice, humanized liver mice, and Sprague-Dawley rats. AMT was identified as a dual substrate and a reversible inhibitor of CYP2D6. Quinidine inhibition of CYP2D6-mediated AMT metabolism showed marked species differences (IC < IC). AMT inhibited MATE1 with an IC of 18.75 μM, with no significant activity against MATE2-K/organic cation transporter 1/2. In humanized liver mice, quinidine coadministration increased AMT exposure (AUCR = 2.87); in rats, AMT coadministration elevated metformin (MATE1 substrate) area under the curve by 68%. Static models predicted that CYP2D6 PMs may have 5- to 10-fold higher AMT exposure, amplifying MATE1 inhibition to increase metformin area under the curve ratio (AUCR) to 2.5-4.1. Coadministration of quinidine was projected to increase metformin AUCR to 2.2. AMT clinical development requires rigorous assessment of the elevated systemic exposure when coadministered with potent CYP2D6 inhibitors. Coadministration with MATE1 substrates (eg, metformin) may delay AMT renal excretion, with superimposed metabolism-transport DDIs posing heightened risk in patients with CYP2D6 PMs or renally impaired. This study highlights the importance of integrating metabolic and transporter pathways for comprehensive DDI risk assessment. SIGNIFICANCE STATEMENT: This study reveals that the novel antidepressant ammoxetine poses dual drug interaction risks as a cytochrome P450 2D6 (CYP2D6) substrate/inhibitor affecting metabolism and a multidrug and toxin extrusion protein 1 inhibitor impairing renal excretion. Using humanized models and static mechanistic models, the interactions were validate in vivo and characterize the proposed metabolism-transport interaction cascade, delineating clinically relevant risks, including amplified ammoxetine exposure with strong CYP2D6 inhibition and heightened vulnerability in CYP2D6 poor metabolizers.